Escherichia coli (E. coli) is a universal cloning host and is the most common organism used in the production of proteins, nucleic acids, metabolites and secondary metabolites in both research and industry. Many of the most important new drugs are manufactured by fermentation in E. coli, which remains the most economical and efficient platform available for production of such molecules. In particular, therapeutics addressing health problems intractable to small-molecule drugs and other traditional treatments. About one-third of all biotherapeutics are currently manufactured in E. coli including single-chain antibodies, which are challenging to develop as well as extremely expensive due in part to limited yields from the E. coli strains in which they are made. Stalled growth is a significant problem that frequently limits E. coli performance and product formation in high density fermentations
E. coli K-12 strains were instrumental in early advances in genetics and microbial physiology and continue to serve as the workhorse of molecular biology methods and techniques. E. coli B strains are often favored as protein expression platforms at laboratory scale, in part due to the availability of strains engineered for use with the T7 polymerase expression systems pioneered by Studier and Moffat [Studier and Moffat, J. Mol. Biol. 189:113 (1986); Studier, J. Mol. Biol. 219:37 (1991)]. At industrial scale, B strains are also recognized as having superior protein production characteristics relative to K-12 strains, even with protein expression systems other than T7 polymerase. Nevertheless, the large quantity of mobile genetic elements, such as insertion sequence elements and prophage, in these strains can cause production problems under stress, particularly the stress encountered when expressing insoluble or toxic targets. On the other hand, E. coli B strains are generally seen as inferior to K strains for producing plasmid DNA.
The phylogenetic relationship of E. coli K and B strains is quite close. To date, more than 60 diverse E. coli strains have been completely or at least substantially sequenced and a detailed phylogenetic map has been produced based on differences among these strains within a series of conserved genes (adk, fumC, icd, gyrB, mdh, purA and recA). All K-12 and B strains fall within a single clade in the E. coli phylogenetic map [Lukjancenko, Wassenaar and Ussery, Microb. Ecol. 60:708 (2010)]. All current K-12 strains are thought to derive from Charles E. Clifton's original K-12 strain (O16, λ+, F+) first isolated at Stanford University in Palo Alto in the late 1920s [Bachman, In Neidhardt, F. C., et al., E. coli and S. typhimurium: Cellular and Molecular Biology. ASM Press, Washington D.C. 1996]. Existing B strains derive largely from Felix d'Herelle's Bacillus coli strain (O7, λR, F−), originally isolated at the Pasteur Institute in Paris as early as 1918 [Daegelen et al., J. Mol Biol 394:634 (2009)]. The close phylogenetic relationship between K-12 and B strains is intriguing from an historical perspective. However, recent advances in our understanding of bacterial evolution and how modern strains may have diverged from ancestral enteric bacteria calls the tidy historical relationship between K and B strains into question [Studier, et al., J. Mol. Biol. 394:653 (2009)]. Regardless, recognition of the differences between K-12 and B strains and their relative advantages for different research and industrial applications is largely based on empirical observation.
The field has long desired a single strain platform possessing superior growth and protein production characteristics of B strains with the genetic tractability and superior DNA production capabilities of K-12 strains. Such an E. coli strain could facilitate development of standard fermentation regimens for optimizing growth and maximizing yield across a diverse spectrum of products.